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Transgenic Animals and Plants

Transgenic Animals and Plants. Genetic Engineering of plant -> Transgenic plants Genetic Engineering of animals -> Transgenic animals. Definition of Transgenic. Transgenic -> stable introduction of a gene into another organism -> For Unicellular organisms (such as bacteria or yeast)

brielle-hendrix
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Transgenic Animals and Plants

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  1. Transgenic Animals and Plants Genetic Engineering of plant -> Transgenic plants Genetic Engineering of animals -> Transgenic animals

  2. Definition of Transgenic Transgenic -> stable introduction of a gene into another organism -> For Unicellular organisms (such as bacteria or yeast) all transformed cells are -> transgenic -> For multicellular organisms (such as animals, plants,..) difference between: - manipulation of single cells -> cell line (expression in insect cells or mammalian cells) - manipulation of a whole plant or animal -> transgenic (can have a transgenic offspring!!!) -> more difficult and expensive to create whole modified organism (transgenic) than just cell line!!!

  3. Transgenic versus Cloning Transgenic -> creation of transgenic animal or plant (introduction of foreign gene into organism) -> transgenic organisms produced by introduction of foreign gene into germ line (-> transgenic offspring!!!) -> introduction of gene into somatic cells -> gene therapy Cloning -> obtaining an organism that is genetically identical to the original organism -> such as Dolly the sheep -> asexual propagation of plants (taking cuttings)

  4. Transgenic Plants Why do we need transgenic plants ? • improvement of agricultural value of plant (resistance to herbicides, resistance to insect attack -> Bacillus thuringiensis toxin) • living bioreactor -> produce specific proteins • studying action of genes during development or other biological processes (knock-out plants, expression down-regulated)

  5. Transgenic Plants • Advantages: • Plant cells are totipotent -> whole plant can be regenerated from a single cell (engineered cells -> engineered plants) • Plants have many offspring -> rare combinations and mutations can be found • Transposons used as vectors • Disadvantages: • Large genomes (polypoid -> presence of many genomes in one cell) • plants regenerating from single cells are not genetically homogenous (genetically instable)

  6. Gene – transfer methods

  7. Agrobacterium tumefaciens mediated transfer

  8. Ti Plasmid

  9. Integration of T-DNA into the plant chromosome -> Tumor formation

  10. Gene transfer by cointegration Recombinant Ti plasmid by recombination

  11. Microprojectile bombardment– “Shotgun”

  12. Viral Vectors

  13. Transfer into protoplasts Vector + polyethylene glycol Gene transfer across a protoplast membrane is promoted by some chemicals such as polyethylene glycol

  14. Electroporation

  15. Control elements on vector Frequently used promoter: -> 35S promoter from cauliflower mosaic virus

  16. Alterations in plant RNA – downregulation of specific genes PG (polygalacturonase) -> Sensitivity of tomatoes to bruising Reduced level-> should give harder fruit during shipping Result: lower level -> did not give harder fruit (more factors responsible for process) Expression of Antisense RNA of transcript of PG -> reduces level of protein produced

  17. Selection marker free transgenic plant-> Transposons

  18. Applications for engineering plants • Development of Insect-, pathogen-, herbicide- resistant plants • Flower pigmentation • Modification of nutritional content • Modification of taste and appearance • Bioreactor • Vaccines (Cholera toxin-like protein in potatoes) • Plant yield (alteration of lignin content -> paper industry)

  19. Development of Insect-, pathogen-, herbicide- resistant plants Toxin from Bacillus thuringiensis

  20. Development of Insect-, pathogen-, herbicide- resistant plants

  21. Development of Insect-, pathogen-, herbicide- resistant plants Manipulations that make a plant herbicide resistance • Inhibit the uptake of the herbicide • overproduce the herbicide-sensitive target protein (Glyphosate) • reduce ability of target protein to bind herbicide (cyclohexanediones) • plant can degrade herbicide (Bromoxynil, Glufosinate, Cyanamide,..)

  22. Development of Insect-, pathogen-, herbicide- resistant plants Fungus- and Bacterium- resistant plants Engineering of plants -> express antimicrobial peptides

  23. Flower pigmentation CHS -> Chalone synthetase -> enzyme in biosynthetic pathway of a purple pigment

  24. Changed nutrition content • Amino acids (to increase lysine content in the future in animal food) • Lipids (possible to change degree of unsaturation, chain length) • Vitamins (Vitamin E, increase Vitamin A in rice)

  25. Modification of taste and appearance Engineer potatoes -> produce more glucose and fructose at higher temperatures

  26. Plants as bioreactor • Therapeutic agents • Antibodies • polymers (PHB)

  27. Transgenic Animals Transgene -> Gene coding for a growth hormone

  28. Transgenic Animals Why do we need transgenic animals ? • living bioreactor -> produce specific proteins in the milk (cattle, sheep, goats, pigs) • studying action of genes during development or other biological processes (knock-out animals, expression down-regulated) -> models for studying human diseases -> mice • improvement of agricultural value (fish, bird)

  29. Gene-transfer methods • Microinjection • Retroviral method • Engineered Embryonic Stem Cells (ES) method • Knock – out methods (Cre-LoxP system) -> studying gene expression + development

  30. The first days of an embryo Used for retroviral infection Fertilized egg Embryonic stem cells (ES)

  31. Microinjection into the germ line -> transgenic animal Gene injected into the male pronuclei

  32. Efficiency of the transgenesis process after DNA microinjection

  33. Retroviral vectors into the germ line (8-cell embryo infected) -> transgenic animal

  34. Engineered Embryonic Stem Cells (ES) into the germ line (blastocyst) -> transgenic animal Engineered ES -> can form any kind of cell in an embryo Inner cell mass (ICM) of blastocysts can form all cells of the embryo -> Pluripotent -> Embryonic stem cells

  35. Gene Therapy – Viral gene transfer into somatic cells Gene transfer into somatic stem cells -> gene therapy Gene transfer via Virus Target tissues: Bone marrow, liver, brain,....

  36. Gene Therapy – Viral gene transfer into somatic cells Gene transfer into somatic stem cells -> gene therapy Used for treating -> genetic diseases, such as diabetes, cancer, color blindness… Different delivery methods

  37. Gene Transfer - what happens on DNA level • Integration into chromosome -> Recombinantion • Recombinantion can be -> homologous – non-homologous • non-homologous event -> more frequently • homologous event -> less frequent but desired • Knock-out mutants -> disrupt functional gene by integration of another gene into target gene • Used for: • -> study human diseases by creating model organisms • -> make minus mutant

  38. Homologous recombinantion

  39. How do check for homologous recombinantion

  40. Construction of a disruption construct

  41. Cre-LoxP system: • Inactivation of a gene (knock-out) in a specific cell type • Activation of a transgene in specific cell type • Used for: • Study biological consequences of tissue- specific gene inactivation • -> establishing models for human diseases • -> selective removal of kinesin II gene (expressed in retinal receptor cells) • -> leads to accumulation of opsin and arrestin -> cell death • -> result mimics aspects of a disease (inherited retinis pigmentosa) • -> large deletions in chromosome -> deletion in chr. 22 -> DiGeorge syndrome • (cardiovascular dysfunction)

  42. Inactivation of gene in specific cell type (tissue)

  43. Cloning of Dolly– Cloning Animals by Nuclear Transfer Technology Critical for success: Cell cycle of the somatic cells (udder cells) on plates was critical – they were kept in specific growth stage (diploid stage) Of the 434 fused oocytes created during the experiment -> only Dolly survived to adulthood Dolly was real clone (genotype identical) and could reproduce Dolly was euthanized 2003 -> suffering from progressive lung disease Since 1997 -> cloning of sheep, cows, mice, cats, other animals done -> many of the clones developed severe diseases as they matured. Until 1997, arrival of Dolly – not possible to produce an adult animal from a nucleus from an adult animal´s differentiated cell

  44. Cloning of Mammals – Reproductive Cloning • Genotype identical • Phenotype is not necessarily identical -> variation due to random events and due to environment

  45. Why do clones have health problems? Telomeres are found at the end of each chromosome. Shrinking of the telomeric ends of our chromosomes are a sign of aging of the cell. Each cycle of cell division the telomeres are slightly shortened until they are too short for further replication -> cell death Dolly´s telomeres (at the age of 3) have been as short as ones of the age of 6 -> clones age “faster”.

  46. Why do clones have health problems? Differentiated cells have certain methylation pattern. Cloned animals have abnormal methylation pattern originating from nucleus from differentiated cells Some can be “re-set” (epigenetic reprogramming) to their undifferentiated state, some cannot -> faulty gene activation in cloned animal -> so few cloned embryos survive -> surviving clones have severe health problems

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